|
| Status |
Public on Feb 26, 2020 |
| Title |
Patient 6 mUM 002 |
| Sample type |
RNA |
| |
|
| Source name |
metastatic Uveal Melanoma Tumor
|
| Organism |
Homo sapiens |
| Characteristics |
individual: Patient 6
tissue: metastatic Uveal Melanoma Tumor
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Formalin fixed paraffin embedded (FFPE) samples were used for total RNA immune gene expression analysis. The tumor areas were selected for RNA extraction, or the entire normal liver tissue. Tissue dissociation, RNA extraction and purification was performed using the RNeasy FFPE Kit (Qiagen) according to manufactures instructions (Qiagen). At least 4 FFPE sections were vortexed in 1ml xylene to remove paraffin wax. Tissues were digested using proteinase K for 15 min at 56 °C and then 15 min at 80 °C. RNA was precipitated and further purified in a RNeasy MinElute spin columns. RNA concentrations were determined using a Qubit® RNA Broad-Range assay. The expression of ~730 immune-related genes were assessed using the NanoString PanCancer Immune Profiling gene set.
|
| Label |
Molecular barcode on reporter probe
|
| Label protocol |
NanoString protocol
|
| |
|
| Hybridization protocol |
Sample hybridization was performed using CodeSet and Capture ProbeSet reagents. Aliquots were used to perform a master mix by adding 70 μL of hybridization buffer to the tube containing the Reporter CodeSet. RNase-free water was added to this mix that the volume of the individual RNA samples was less than 5 μL. 8 μL of master mix was added to each of the 11 tubes. 5 μl of sample was added to each tube. 2 μL of Capture ProbeSet was added to each tube immediately before placing at 65°C. Reactions was performed for at least 16 hours, further stopped at 4°C and processed in the following day.
|
| Scan protocol |
After hybridization, excess probes are washed away and the Capture Probes and Target/Probe Complexes are eluted off of the beads and are then hybridized to magnetic beads complementary to the Reporter Probe. Wash steps are performed and excess Capture Probes are washed away. Finally, the purified Target/Probe Complexes are eluted off and are immobilized in the cartridge for data collection. Data collection is carried out in the nCounter® Digital Analyzer. Fields of view (FOV) are collected per sample using a microscope objective and a CCD camera yielding data of hundreds of thousands of target molecule counts. Digital images are processed on the Digital Analyzer and the barcode counts are then tabulated and displayed.
|
| Data processing |
The NanoString nSolver 2.6 software was used for normalization of expression counts using housekeeping genes following the manufacturer’s recommendations (NanoString). normalized within each immune category (e.g. CTLs suppression, M2 macrophages regulation and Immune checkpoint regulators).
Normalized signal intensity. Data are displayed in expression count units of individual gene per patient and normal liver sample.
|
| |
|
| Submission date |
Feb 23, 2020 |
| Last update date |
Feb 27, 2020 |
| Contact name |
Carlos Rogerio De Figueiredo |
| E-mail(s) |
[email protected]
|
| Organization name |
University of Liverpool
|
| Department |
Molecular and Clinical Cancer Medicine
|
| Lab |
LOORG
|
| Street address |
6 West Derby Street
|
| City |
Liverpool |
| State/province |
Merseyside |
| ZIP/Postal code |
L7 8TX |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL25262 |
| Series (1) |
| GSE145782 |
Transcriptomic analysis of BAP1 negative primary and metastatic uveal melanoma tumors |
|
